Multi donor stem cell compositions and methods of making same

ABSTRACT

Disclosed are compositions, in particular, organoid compositions, derived from more than one donor cell. Further disclosed are methods of making compositions, for example, organoid compositions, that comprise a differentiated cell population derived from more than one donor cell. Donor cells may include, for example, a precursor cell such as an embryonic stem cell or other precursor cell. The disclosed methods use synchronization conditions to produce a synchronized pooled-precursor cell population, which may then be differentiated into an organoid composition. Methods of using the compositions are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalApplication 62/485,562 filed Apr. 14, 2017, and U.S. ProvisionalApplication 62/517,414 filed Jun. 9, 2017. The contents of each areincorporated in their entirety for all purposes.

BACKGROUND

Efficient methods for the study of large populations are an unmet needin the art. For example, the study of safety and efficacy of a potentialnew drug or treatment in a population is a requirement for clinicaltrials for drug approval. Preclinical detection of risk compounds fordrug induced liver injury (DILI) remain a significant challenge in drugdevelopment, highlighting a need for a predictive human system. That is,clinical trials are costly and time consuming, and many drugs fail fortoxicity and/or lack of efficacy—often after much investment in clinicaltrials has been made. Early determination of toxicity (in particular,drug induced hepatotoxicity) or efficacy using in vitro research toolsthat are representative of a population could shorten the time todevelopment and/or avoid unnecessary injury to clinical trialparticipants, and/or decrease costs associated with bringing drugs tomarket. A viable in vitro platform to screen a population cohort fordrug safety and efficacy studies would therefore be highly desirable.Further desirable is a screen useful for early detection in vulnerablepopulations, such as individuals having compromised liver function.

In addition, there exists many disease states for which a genetic causeis unknown—there is therefore a need for improved methods foridentifying the genetic basis for disease states that appear in apopulation. Human induced pluripotent stem cells (hiPSC) and otherprecursor cells provide a promising opportunity to establishhuman-specific models of various disease for drug development.Population-scale iPSC reprogramming initiatives may allow for the studyof any imaginable population cohort for disease phenotypes, drug safety,and efficacy. However, one to one comparison of each individual stemcell is inefficient and completely unrealistic due to both time and costconsiderations. Therefore, a viable platform to screen a populationphenotype in a pooled sample of various donor derived precursor cellswould be highly desirable. While methods of making differentiated cellpopulations and organoid compositions from precursor cells have beendescribed in the art, directed differentiation of cell populationsand/or organoids derived from more than one donor has not beenaccomplished due to the need to synchronize growth and differentiationof cells from different donors. The instant disclosure seeks to addressone or more of the aforementioned needs in the art.

BRIEF SUMMARY

Disclosed are compositions, in particular, organoid compositions,derived from more than one donor cell, thus allowing for cellularrepresentation of one or more donors, or, in some cases, a population ofdonors. Further disclosed are methods of making such compositions, forexample, organoid compositions that comprise a differentiated cellpopulation derived from more than one donor cell that may represent adesired population of donors. Donor cells useful for deriving thedisclosed organoid compositions may include, for example, a precursorcell such as an embryonic stem cell or other precursor cell. Thedisclosed methods use synchronization conditions to produce asynchronized pooled-precursor cell population, which may then bedifferentiated via directed differentiation into an organoidcomposition. Methods of using the compositions are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

This application file contains at least one drawing executed in color.Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1 depicts an analysis of cell proliferation and metabolism toexamine donor-dependent variation. Top: Cell proliferation assay, whichshows the difference in the proliferative property of iPSCs. iPSCs wereplated at 5×10 4 cells and cell numbers were count. Bottom: Metabolomicsanalysis showing the difference in metabolism of iPSCs. PCA scores wereplotted using the 30 metabolites assigned buckets.

FIG. 2 shows synchronizing growth kinetic variabilities by hypoxicculture condition. Hypoxic culture promoted cell synchronization byregulating cell growth kinetics. Top: Morphology of cells in hypoxicculture condition. Bottom left: Cell proliferation in each oxygenconditioning. Cell proliferation of donor-dependent variation wascanceled in the 2% hypoxic condition. Bottom right: Cell proliferationsynchronization analysis. The difference in the growth rate of each cellwas plotted. Synchronization index (SYNDEX) is the reciprocal of donordifference standard deviation.

FIG. 3 depicts synchronizing growth kinetic variabilities by compoundcocktail treatment. Compound cocktail treatment was supported cellsynchronization by combining with hypoxic culture. Top: Cellproliferation in inhibitor treatment. Bottom: Cell proliferationsynchronization analysis.

FIG. 4 shows pooling culture experiment of iPSC derived from multidonors. Multiple iPSCs with fluorescent labels were mixed and culturedin a hypoxic state. As a result of fluorescence microscopic observationand FACS population analysis, hypoxic condition enabled synchronizationand maintenance in each cell population on the same dish.

FIG. 5 shows parallel differentiation induction of various cell typesfrom iPSC pool. In order to confirm the pluripotency of pooled iPSC,Applicant attempted to induce multiple lineage including definitiveendoderm, mesoderm, posterior foregut, endothelial cells, hepatocytes,and liver organoids. As a result of gene expression, FACS analysis andspecific functional analysis, it was demonstrated that pooled iPSC hadmulti lineage differentiation potential.

FIG. 6 shows iPSC pooling overcomes differentiation propensity toendothelial cells. In order to confirm the effect of iPSC pooling,Applicant attempted to induce endothelial cell induction using iPSClines with low endothelial induction efficiency. Top: The morphology ofendothelial cell induction in each cell line and pooled cell line.Bottom: FACS analysis of endothelial specific marker expression in eachcell line and pooled cell line. The endothelial differentiationefficiency was significantly improved in pooled iPSC culture.

FIG. 7 shows confirmation of neural differentiation induction efficiencyof iPSC established from multiple donor-derived fibroblast. Neuriteoutgrowth was observed on day 23 of induction of neuronaldifferentiation of multiple donor-derived iPSC

FIG. 8 shows confirmation of neural differentiation induction efficiencyof iPSC established from multiple donor-derived fibroblast. Expressionof neuron-specific genes were significantly increased at day 23 ofdifferentiation induction.

FIG. 9 shows confirmation of neural differentiation induction efficiencyof iPSC established from multiple donor-derived fibroblast. The neuronspecific markers TUJ 1, PAX 6 and TH were expressed at day 23 ofdifferentiation.

FIG. 10 shows confirmation of neural differentiation inductionefficiency of iPSC established from multiple donor-derived fibroblast.The neuron specific markers TUJ 1, PAX 6 and TH were expressed at day 23of differentiation.

FIG. 11 shows establishment of freezing method of iPSC derived foregutcell. To establish the freezing method of iPSC-derived foregut cell, weattempted several cryopreservation conditions. As a result of freezingand thawing, the survival rate of single cell isolated cells wassignificantly higher than the cluster condition cell.

FIG. 12 shows establishment of freezing method of iPSC derived foregutcell. To establish the freezing method of iPSC-derived foregut cell,Applicant attempted to generate liver organoids using frozen cells. As aresult of organoid formation, the organoid formation efficiency ofsingle cell isolated cells was significantly higher than the clustercondition cell.

FIG. 13 shows clonal liver organoid formation from frozen foregut pool.Applicant developed a method to form liver organoids from frozen foregutpools. Organoid formation was performed by plating 5000 cells to 40000cells in 10 μl Matrigel drops, respectively. The pooled cells were usedfluorescently labeled foregut cells (Green, Red, blue) and non-labeledforegut cells.

FIG. 14 shows clonal liver organoid formation from iPSC pool. Organoidswere successfully formed under the conditions of 20000 cells and 40000cells. In addition, it was confirmed that the formed organoid wasmonochromatic and derived from a single donor.

FIG. 15 shows live imaging-based detection of iron accumulation inhepatocyte and organoids from pool. The pooled cells were usedfluorescently labeled foregut cells (Green, blue) and non-labeledforegut cells. For the detection of iron accumulation, a red fluorescentreagent FeRhoNox-1 which reacts specifically with iron ions was used.

FIG. 16 shows live imaging-based detection of iron accumulation inhepatocyte and organoids from pool. Two types of fluorescently labeledcells and diseased cells derived from hemochromatosis were used as apositive control for iron accumulation to prepare pooled hepatocytes.Quantitative results of iron accumulation show that it was significantlyhigher in hemochromatosis derived cells.

FIG. 17 shows live imaging-based detection of iron accumulation inhepatocyte and organoids from pool. As with 2D hepatocyte experiment,two types of fluorescently labeled cells and hemochromatosis derivedcells were used to prepare pooled liver organoids. Quantitative resultsof iron accumulation were significantly higher in cells derived fromhemochromatosis.

FIG. 18 shows development of non-invasive donor decoding method inpooled stem cell culture by environmental DNA and a conceptual image ofdonor identification using donor specific SNPs PCR.

FIG. 19 shows development of non-invasive donor decoding method inpooled stem cell culture by environmental DNA and a conceptual image ofdonor identification using donor specific SNPs PCR.

FIG. 20 shows donor ratio estimation from culture medium in iPSC pool.Applicant attempted to estimate the donor ratio of iPSC pool usingenvironmental DNA (“eDNA”) contained in the culture supernatant. As aresult, the cell ratio was successfully identified by PCR of eDNA inculture medium. The eDNA ratio in the culture medium was also correlatedwith attached cells.

FIG. 21 shows donor ratio estimation from culture medium in iPSC pool.Applicant also attempted to estimate the donor ratio of pooledhepatocyte and liver organoid by eDNA. As a result, the cell ratio wassuccessfully identified by PCR of eDNA in the medium.

FIG. 22. Donor ratio change monitored by gDNA in the culture medium. Toconfirm the eDNA donor identification, Applicant monitored cell ratiochange by drug selection. Two types of fluorescently labeled cells andpuromycin resistant cells were used as a positive control. As a resultof drug selection, non-resistant iPSCs were disappeared on the secondday of treatment. The result of eDNA donor identification PCR showedthat the ratio of nonresistant cells decreased from the second day andonly resistant cells were detected on the fourth day.

FIG. 23. Drop-out assay using induced hepatocytes treated with oleicacid and troglitazone. Drug screening assays for fat accumulation anddrug treatment were performed using hepatocytes prepared from iPSCpools. Eleven iPSCs with different donors were used for iPSC derivedhepatocyte pool. eDNA based drop-out assay confirmed cells with high fataccumulation risk donor were significantly more sensitive to druginduced hepatotoxicity than healthy donor derived cells

DETAILED DESCRIPTION Definitions

Unless otherwise noted, terms are to be understood according toconventional usage by those of ordinary skill in the relevant art. Incase of conflict, the present document, including definitions, willcontrol. Preferred methods and materials are described below, althoughmethods and materials similar or equivalent to those described hereincan be used in practice or testing of the present invention. Allpublications, patent applications, patents and other referencesmentioned herein are incorporated by reference in their entirety. Thematerials, methods, and examples disclosed herein are illustrative onlyand not intended to be limiting.

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a method” includesa plurality of such methods and reference to “a dose” includes referenceto one or more doses and equivalents thereof known to those skilled inthe art, and so forth.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, or up to 10%, or up to 5%, or up to 1% of a given value.Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, for examplewithin 5-fold, or within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably to refer to an animal that is the object of treatment,observation and/or experiment. Generally, the term refers to a humanpatient, but the methods and compositions may be equally applicable tonon-human subjects such as other mammals. In some embodiments, the termsrefer to humans. In further embodiments, the terms may refer tochildren.

As used herein, the term “pluripotent stem cells (PSCs)” encompasses anycells that can differentiate into nearly all cell types of the body,i.e., cells derived from any of the three germ layers (germinalepithelium), including endoderm (interior stomach lining,gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood,urogenital), and ectoderm (epidermal tissues and nervous system). PSCscan be the descendants of inner cell mass cells of the preimplantationblastocyst or obtained through induction of a non-pluripotent cell, suchas an adult somatic cell, by forcing the expression of certain genes.Pluripotent stem cells can be derived from any suitable source. Examplesof sources of pluripotent stem cells include mammalian sources,including human, rodent, porcine, and bovine.

As used herein, the term “induced pluripotent stem cells (iPSCs),” alsocommonly abbreviated as iPS cells, refers to a type of pluripotent stemcells artificially derived from a normally non-pluripotent cell, such asan adult somatic cell, by inducing a “forced” expression of certaingenes. hiPSC refers to human iPSCs. In some embodiments, iPSCs may bederived by transfection of certain stem cell-associated genes intonon-pluripotent cells, such as adult fibroblasts. Transfection may beachieved through viral vectors, such as retroviruses. Transfected genesmay include the master transcriptional regulators Oct-3/4 (Pouf51) andSox2, although other genes may enhance the efficiency of induction.After 3-4 weeks, small numbers of transfected cells begin to becomemorphologically and biochemically similar to pluripotent stem cells, andare typically isolated through morphological selection, doubling time,or through a reporter gene and antibiotic selection. As used herein,iPSCs include first generation iPSCs, second generation iPSCs in mice,and human induced pluripotent stem cells. In some embodiments, aretroviral system is used to transform human fibroblasts intopluripotentstem cells using four pivotal genes: Oct3/4, Sox2, Klf4, and c-Myc. Inalternative embodiments, a lentiviral system is used to transformsomatic cells with OCT4, SOX2, NANOG, and LIN28. Genes whose expressionare induced in iPSCs include but are not limited to Oct-3/4 (e.g.,Pou5fl); certain members of the Sox gene family (e.g., Sox1, Sox2, Sox3,and Sox15); certain members of the Klf family (e.g., Klf1, Klf2, Klf4,and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, andN-myc), Nanog, and LIN28.

As used herein, the term “embryonic stem cells (ESCs),” also commonlyabbreviated as ES cells, refers to cells that are pluripotent andderived from the inner cell mass of the blastocyst, an early-stageembryo. For purpose of the present invention, the term “ESCs” is usedbroadly sometimes to encompass the embryonic germ cells as well.

As used herein, the term “precursor cell” encompasses any cells that canbe used in methods described herein, through which one or more precursorcells acquire the ability to renew itself or differentiate into one ormore specialized cell types. In some embodiments, a precursor cell ispluripotent or has the capacity to becoming pluripotent. In someembodiments, the precursor cells are subjected to the treatment ofexternal factors (e.g., growth factors) to acquire pluripotency. In someembodiments, a precursor cell can be a totipotent (or omnipotent) stemcell; a pluripotent stem cell (induced or non-induced); a multipotentstem cell; an oligopotent stem cells and a unipotent stem cell. In someembodiments, a precursor cell can be from an embryo, an infant, a child,or an adult. In some embodiments, a precursor cell can be a somatic cellsubject to treatment such that pluripotency is conferred via geneticmanipulation or protein/peptide treatment. Precursor cells includeembryonic stem cells (ESC), embryonic carcinoma cells (ECs), andepiblast stem cells (EpiSC).

Applicant has developed a system for synchronization of growth kineticvariabilities among multiple iPSC and expansion and differentiation ofstem cell pool into specific cell types, which may be further developedinto an organoid of a desired type, but which may comprise cells frommore than one donor. In developmental biology, cellular differentiationis the process by which a less specialized cell becomes a morespecialized cell type. As used herein, the term “directeddifferentiation”, or more generically, “differentiation” in situationsin which external factors are applied to induce or controldifferentiation, may describe a process through which a less specializedcell becomes a particular specialized target cell type. Theparticularity of the specialized target cell type can be determined byany applicable methods that can be used to define or alter the destinyof the initial cell. Exemplary methods include but are not limited togenetic manipulation, chemical treatment, protein treatment, and nucleicacid treatment.

In one aspect, a composition comprising a differentiated cell populationderived from more than one precursor cell is disclosed, wherein saiddifferentiated cell population is synchronized in development—i.e., thecells are differentiated to a desired cellular stage at substantiallythe same point in time, for example, via mitotically promoting slowcycling stem cells and/or inhibiting fast cycling stem cells. Thesynchronized, differentiated cell population may comprise cells frommore than one individual. In one aspect, the synchronized,differentiated cell population may be selected from definitive endoderm,mesoderm, exoderm, posterior foregut, endothelium, hepatocytes, or anorganoid. The more than one individual may, in certain aspects, comprisea population of individuals with or without a disease of interest.

In some embodiments, stem cells that are pluripotent or can be inducedto become pluripotent. In some embodiments, pluripotent stem cells arederived from embryonic stem cells, which are in turn derived fromtotipotent cells of the early mammalian embryo and are capable ofunlimited, undifferentiated proliferation in vitro. Embryonic stem cellsare pluripotent stem cells derived from the inner cell mass of theblastocyst, an early-stage embryo. Methods for deriving embryonic stemcells from blastocytes are well known in the art. Human embryonic stemcells H9 (H9-hESCs) are used in the exemplary embodiments described inthe present application, but it would be understood by one of skill inthe art that the methods and systems described herein are applicable toany stem cells.

Additional stem cells that can be used in embodiments in accordance withthe present invention include but are not limited to those provided byor described in the database hosted by the National Stem Cell Bank(NSCB), Human Embryonic Stem Cell Research Center at the University ofCalifornia, San Francisco (UCSF); WISC cell Bank at the Wi Cell ResearchInstitute; the University of Wisconsin Stem Cell and RegenerativeMedicine Center (UW-SCRMC); Novocell, Inc. (San Diego, Calif.);Cellartis AB (Goteborg, Sweden); ES Cell International Pte Ltd(Singapore); Technion at the Israel Institute of Technology (Haifa,Israel); and the Stem Cell Database hosted by Princeton University andthe University of Pennsylvania. Exemplary embryonic stem cells that canbe used in embodiments in accordance with the present invention includebut are not limited to SA01 (SA001); SA02 (SA002); ES01 (HES-1); ES02(HES-2); ES03 (HES-3); ES04 (HES-4); ES05 (HES-5); ES06 (HES-6); BG01(BGN-01); BG02 (BGN-02); BG03 (BGN-03); TE03 (13); TE04 (14); TE06 (16);UC01 (HSF1); UC06 (HSF6); WA01 (H1); WA07 (H7); WA09 (H9); WA13 (H13);WA14 (H14). More details on embryonic stem cells can be found in, forexample, Thomson et al., 1998, “Embryonic Stem Cell Lines Derived fromHuman Blastocysts,” Science 282 (5391):1145-1147; Andrews et al., 2005,“Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: oppositesides of the same coin,” Biochem Soc Trans 33:1526-1530; Martin 1980,“Teratocarcinomas and mammalian embryogenesis,” Science 209(4458):768-776; Evans and Kaufman, 1981, “Establishment in culture ofpluripotent cells from mouse embryos,” Nature 292 (5819): 154-156;Klimanskaya et al., 2005, “Human embryonic stem cells derived withoutfeeder cells,” Lancet 365 (9471): 1636-1641; each of which is herebyincorporated herein in its entirety.

In one aspect, the differentiated cell population may comprise anorganoid, the organoid being derived from a precursor cell from morethan one donor, or more than one to about 1000 donors, or from about 100donors to about 500 donors. The organoid may be any organoid known toone of ordinary skill in the art, and may include, for example, a liverorganoid, a gastric organoid, an intestinal organoid, a brain organoid,a pulmonary organoid, a bone organoid, a cartilage organoid, a bladderorganoid, a blood vessel organoid, an endocrine organoid (such asthyroid, pituitary, adrenal, or the like), a sensory organoid (ear, eye,taste, tongue, or the like).

Methods of developing organoid tissues have been disclosed in the art.The disclosed methods allow for development of an organoid derived froma precursor cell, such as an iPSC, or embryonic stem cell (ES cell),from more than one individual. The resulting “pooled organoid” combinescells from more than one donor, providing unique properties to andallowing for novel uses for the resulting organoid. Methods of makingvarious organoids are known in the art and are described in, forexample, the following references: Dekkers, J. F., Wiegerinck, C. L., deJonge, H. R., Bronsveld, I., Janssens, H. M., de Winter-de Groot, K. M.,Brandsma, A. M., de Jong, N. W., Bijvelds, M. J., Scholte, B. J., et al.(2013). A functional CFTR assay using primary cystic fibrosis intestinalorganoids. Nat Med 19, 939-945; Lancaster, M. A., and Knoblich, J. A.(2014). Organogenesis in a dish: modeling development and disease usingorganoid technologies. Science 345, 1247125; Nakamura, T., and Sato, T.(2018). Advancing Intestinal Organoid Technology Toward RegenerativeMedicine. Cell Mol Gastroenterol Hepatol 5, 51-60; Sachs, N., de Ligt,J., Kopper, O., Gogola, E., Bounova, G., Weeber, F., Balgobind, A. V.,Wind, K., Gracanin, A., Begthel, H., et al. (2018). A Living Biobank ofBreast Cancer Organoids Captures Disease Heterogeneity. Cell 172,373-386 e310; Sasai, Y. (2013). Cytosystems dynamics inself-organization of tissue architecture. Nature 493, 318-326; Takebe,T., Sekine, K., Enomura, M., Koike, H., Kimura, M., Ogaeri, T., Zhang,R. R., Ueno, Y., Zheng, Y. W., Koike, N., et al. (2013). Vascularizedand functional human liver from an iPSC-derived organ bud transplant.Nature 499, 481-484; Takebe, T., Sekine, K., Kimura, M., Yoshizawa, E.,Ayano, S., Koido, M., Funayama, S., Nakanishi, N., Hisai, T., Kobayashi,T., et al. (2017). Massive and Reproducible Production of Liver BudsEntirely from Human Pluripotent Stem Cells. Cell Rep 21, 2661-2670;Workman, M. J., Mahe, M. M., Trisno, S., Poling, H. M., Watson, C. L.,Sundaram, N., Chang, C. F., Schiesser, J., Aubert, P., Stanley, E. G.,et al. (2017). Engineered human pluripotent-stem-cell-derived intestinaltissues with a functional enteric nervous system. Nat Med 23, 49-59.

In one aspect, a method of making a composition comprising adifferentiated cell population derived from a plurality of donors isdisclosed. The differentiated cell population may comprise a populationof cells derived from more than one donor, for example, from about 10donors to about 1,000 donors or more, or from about 100 to about 500donors. The method may comprise the steps of exposing a pooled-iPSCpopulation to a synchronization condition for a period of timesufficient to produce a synchronized pooled-iPSC population; contactingthe synchronized pooled-iPSC population with a “compound cocktail”sufficient to differentiate the synchronized pooled-iPSC population intoa differentiated cell population. In one aspect, the compound cocktailmay comprise a mitogen-activated protein kinase inhibitor (MEK/ERKpathway inhibitor), for example, PD0325901 and a glycogen synthasekinase-3 inhibitor, for example, CHIR99021. In other aspects, thecompound cocktail may further comprise an estrogen-related receptorgamma agonist, for example, GSK5182, a Rho-associated kinase inhibitor(Rho/ROCK pathway inhibitor), for example Y-27632 and a type 1transforming growth factor-b receptor inhibitor, for example A-83-01.Exemplary concentrations include GSK5182; 5 nM-50 μM, PD0325901; 1 nM-10μM, CHIR99021; 3 nM-30 μM, A83-01; 0.5 nM-5 μM, Y27632; 10 nM-100 μM

In other aspects, the following agents may be used in lieu of or inaddition to the aforementioned agents.

RHO/ROCK pathway inhibitor: Y-27632 dihydrochloride (R&D #1254)

GSK3 pathway inhibitors: CHIR 99021(R&D #4423), Beryllium, Copper,Lithium, Mercury, Tungsten, 6-BIO, Dibromocantharelline, Hymenialdesine(Enzo Life Sciences), Indirubins, Meridianins, CT98014 (Axon Medchem),CT98023 (BOC Sciences), CT99021, TWS119, SB-216763, SB-41528,AR-A014418, AZD-1080, Alsterpaullone (Sigma), Cazpaullone (MedKoo),Kenpaullone (Sigma), Manzamine A(Enzo Life Sciences), Palinurine,Tricantine, TDZD-8, NP00111, NP031115, Tideglusib, HMK-32, L803-mts(Sigma), Ketamine (Sigma), or combinations thereof. Such agent may bepresent in an amount of from about 10 nM to about 100 μM, or from about100 nM to about 50 μM.

TGFB pathway inhibitors: Stemolecule A83-01(Stemgent #47132), SD 208,LY2109761, SB525334, Pirfenidone, GW788388, RepSox, LY 2157299, LDN193189 hydrochloride, SB 431542, Dorsomorphin, A 77-01, GW 788388,Dorsomorphin dihydrochloride, K 02288, SB 505124, LDN 212854trihydrochloride, SB 525334, ITD 1, SIS3, or combinations thereof. Suchagent may be present in an amount of from about 0.5 nM to about 5 μM, orfrom about 1 nM to about 2.5 μM.

MEK/ERK Pathway Inhibitors, PD0325901 (R&D #4192/10) (R&D #4192/10),Selumetinib (AZD6244), PD0325901, Trametinib (GSK1120212), U0126-EtOH,PD184352 (CI-1040), PD98059, BIX 02189, Pimasertib (AS-703026), BIX02188, TAK-733, AZD8330, Binimetinib (MEK162, ARRY-162, ARRY-438162),PD318088, Honokiol, SL-327, Refametinib (RDEA119, Bay 86-9766),Myricetin, BI-847325, Cobimetinib (GDC-0973, RG7420), APS-2-79 HC1,GDC-0623, or combinations thereof. Such agent may be present in anamount of from about 1 nM to about 10 μM, or from about 10 nM to about 5μM.

Estrogen-related receptor γ (ERRγ) agonists: GS K5182 (Aobious#AOB1629), AC 186, Daidzein, DPN, (R)-DPN, DY131, (S)-Equol, ERB 041,α-Estradiol, β-Estradiol, Estropipate, FERb 033, GSK 4716,27-Hydroxycholesterol, Liquiritigenin, PPT, WAY 200070, XCT 790, orcombinations thereof. Such agent may be present in an amount of fromabout 5 nM to about 50 μM, or from about 10 nM to about 25 μM.

Source information: The following may be obtained, for example, fromSelleck Chemical company: Indirubins, 6-BIO, CT99021, TWS119, SB-216763,SB-41528, AR-A014418, AZD-1080, TDZD-8, Tideglusib, SD 208, LY2109761,SB525334, Pirfenidone, GW788388, RepSox, LY 2157299, LDN 193189hydrochloride, SB 431542, Dorsomorphin, A 77-01, GW 788388, Dorsomorphindihydrochloride, K 02288, SB 505124, LDN 212854 trihydrochloride, SB525334, ITD 1, SIS3, Selumetinib (AZD6244), Trametinib (GSK1120212),U0126-EtOH, PD184352 (CI-1040), PD98059, BIX 02189, Pimasertib(AS-703026), BIX 02188, TAK-733, AZD8330, Binimetinib (MEK162, ARRY-162,ARRY-438162), PD318088, Honokiol, SL-327, Refametinib (RDEA119, Bay86-9766), Myricetin, BI-847325, Cobimetinib (GDC-0973, RG7420), APS-2-79HCl GDC-0623, AC 186, Daidzein, DPN, (R)-DPN, DY131, (S)-Equol, ERB 041,α-Estradiol, β-Estradiol, Estropipate, FERb 033, GSK 4716,27-Hydroxycholesterol, Liquiritigenin, PPT, WAY 200070, XCT 790.

In one aspect, the cells are in culture for at least four days to allowfor synchronization of the mixed donor cell population. In otheraspects, the amount of time for synchronization may be from about 1 toabout 6 or about 2 to about 5 or about 4 days. For proliferativekinetics, eDNA based monitoring of non-labeled stem cell pools orimage-based analysis of differently labeled stem cell pools can be used.In one aspect, the methods may be carried out for a period of timesufficient for the following markers of pluripotentcy to be expressed:NANOG, OCT4, SOX2, SSEA4 and TRA160. In one aspect, the synchronizationcondition may comprise a hypoxic culture condition, for example, whereinsaid hypoxic culture condition is between about 2% 02 to about 10% O₂,or between about 2% to about 5%, or about 2%, or about 3%, or about 4%,or about 5%, or up to 6% or up to 7%, or up to about 8% O₂

In one aspect, a method of making a composition comprising adifferentiated cell population from more than one donor is disclosed.The differentiated cell population may be derived from more than oneindividual, for example, from about 10 individuals to about 1,000donors, or from about 100 to about 500 donors. The method may comprisethe steps of exposing a pooled-endoderm population to a clonal conditionfor a period of time sufficient to produce a synchronizedpooled-endoderm population; contacting said synchronized pooled-endodermpopulation with a compound cocktail sufficient to differentiate saidsynchronized pooled-endoderm population into a differentiated cellpopulation, wherein said compound cocktail comprises, for example a TGFBreceptor pathway inhibitor, for example, A-83-01, a glycogen synthasekinase-3 (GSK3) pathway inhibitor, for example, CHIR99021, FGF pathwaystimulant, for example, FGF2, EGF pathway stimulant, for example, EGF,and VEGF pathway stimulant, for example, VEGF. In one aspect, theendoderm may comprise posterior foregut cells.

An endoderm population is intended to encompass one or more endodermcells. Likewise, posterior foregut cells and a population thereof isintended to encompass one or more posterior foregut cells. Both endodermcells and posterior foregut cells can be obtained using methods known inthe art, for example, as described in Cheng, Xin et al. “Self-RenewingEndodermal Progenitor Lines Generated from Human Pluripotent StemCells.” Cell Stem Cell, Volume 10, Issue 4, 371-384; Hannan, Nicholas R.F. et al., “Generation of Multipotent Foregut Stem Cells from HumanPluripotent Stem Cells” Stem Cell Reports, Volume 1, Issue 4, 293-306;and Zhang, Ran-Ran et al. “Human iPSC-Derived Posterior Gut ProgenitorsAre Expandable and Capable of Forming Gut and Liver Organoids” Stem CellReports, Volume 10, Issue 3, 780-793

By clonal conditions, it is meant: conditions in which a culture ofcells may be produced in such a way that an individual clone can beselected. For example, stem cells with multilineage differentiationpotential and self-renewing capability may be clonally propagated in aclonal culture (clonal condition) to continuously produce hepatocytesand cholangiocytes as descendants while maintaining primitive stemcells, as described in Suzuki A, Zheng Y, Kaneko S, et al. Clonalidentification and characterization of self-renewing pluripotent stemcells in the developing liver. The Journal of Cell Biology. 2002;156(1):173-184. doi:10.1083/jcb.200108066. In other aspects, the clonalculture (clonal condition) may include the feature of seeded cells withdecreased density in culture as compared to what is typical in the art.For example, 1000-40000 cells embedded in 10 μl Matrigel will generateover 100-4000 of clone-derived organoids. Using this density, a singledonor derived cells can be isolated, allowing for a single donor derivedorganoid. Clonal cell culture conditions are readily understood by oneof ordinary skill in the art, and variations in protocol will be readilyappreciated.

In one aspect, disclosed is a viable platform for screening a populationphenotype in a pooled sample of various donor derived iPSCs. Humaninduced pluripotent stem cells (hiPSC) and other precursor cells providea promising opportunity to establish human-specific models of variousdisease for drug development. Population-scale iPSC reprogramminginitiatives may allow for the study of any imaginable population cohortfor disease phenotypes, drug safety, and efficacy. However, one to onecomparison of each individual stem cell is inefficient and completelyunrealistic due to both time and cost considerations. The disclosedcompositions and methods further allow for forward identification ofrare individual phenotypes at the single cell level. Using the disclosedcompositions and methods, a phenotype may be screened at a populationscale in a single dish, enabling a new strategy to capture individualphenotype from a pooled sample—an approach termed “forward cellomicsapproach.” Such population scale hiPSC may provide an in vitropopulation cohort for drug safety and efficacy studies, thus addressingthe aforementioned problems in the art.

Preclinical detection of risk compounds for drug induced liver injury(DILI) remain a significant challenge in drug development, highlightinga need for a predictive human system. Applicant has developed liverorganoids from pooled iPSCs for analyzing humanistic DILI pathology atorganoid resolution. Differentiated liver organoids from human iPSCcontain polarized hepatocytes and an internal lumen lined bydifferentiated cells, recapitulating the in vivo bile canaliculi-likearchitecture. Applicant leveraged this structural feature for modelingDILI with multiplexed live imaging, termed “LoT” (Liver organoid-basedToxicity screen). LoT was functionally validated with 10 marketed drugs,distinguishing respective mechanism-of-action based on cholestasis andmitochondrial toxicity. (Data not shown.) Organoids were introduced in avulnerable (steatotic) condition to demonstrate toxicity exaggeration asshown in clinics, followed by chemical rescue from massive organoiddeath. Thus, Applicant has shown the first human organoid-based modelfor drug safety with a cost-effective platform, facilitating compoundoptimization, mechanistic study, and precision medicine as anti-DILItherapy screening applications. Further, current phase 1 or other trialphases completely lack the risk assessment process of vulnerableconditions—the disclosed LoT methods allow use of a vulnerable (e.g.,steatotic) condition to demonstrate toxicity exaggeration and addressesthe huge financial loss that results from drug withdrawal byunpredictable hepatoxicity in current trials.

In one aspect, the disclosed methods and compositions allow forpopulation-scale hiPSC reprogramming, which can then, in turn, allow foran in vitro population cohort for drug safety and efficacy studies. Forexample, a “healthy” or “disease” iPSC bank may be developed using themethods disclosed herein. For example, a single “iPSC bank” may compriseindividual iPSCs that are derived from multiple donors. In someinstances, the number of iPSC donor lines may be from about 100 iPSCdonor lines up to over 1000 iPSC donor lines, or from more than one toabout 500 donor lines. The instant disclosure provides methods by whichthe iPSC donor cells may be successfully synchronized and pooled tocreate a “pooled-iPSC” composition or “bank” of iPSC cells that arederived from more than one individual. The pooled-iPSCs may then besubjected to differentiation methods for the production of an organoid.The organoid may be, for example, a liver organoid (“mini-liver”) fromthe iPSC bank which, because of the ability to synchronize the iPSCcells, contain cells representative of a desired population.

In one aspect, liver organoids derived from the iPSC bank may be usedfor a variety purposes including drug screening and transplantapplication, Phase I studies, including hepatoxicity screening for bothnormal and compromised cells (e.g., steatotic liver cells/organoids),NAFLD/Cholestasis drug discovery screening, nutritional screening (suchas, for example, TPN, supplements), or Phase II studies, nutrition andDILI Precision screening, mini-organ transplants, resilient plasmaproduct generation, or as an FXR agonist (bile acid bearing intra-oralorganoid delivery). In particular, bile acid or its analogue, is knownas an FXR (farnesoid x receptor) agonist, which is currently used fortreatment of NASH and other liver diseases. The disclosed organoids maybe used for production of bile acids, which may be used for treatment ofNASH and other such liver diseases. In other aspects, the organoiditself may be used in vivo as an FXR agonist when orally administeredlike compounds.

In one aspect, a method of testing a compound for a characteristic ofinterest is disclosed. The method may comprise the step of contacting atest composition of interest with the pooled organoid composition asdescribed above composition. The characteristic of interest may beselected from cell death, cell growth, cell viability, cholestasis,mitochondrial overload, ROS production indicative of mitochondrialdysfunction, fibrosis, cell stiffness, fibrosis, efficacy for a diseasestate, and combinations thereof. The test compound may be selected froma drug-like molecule, a therapeutic agent, a nutritional supplement, orcombinations thereof. In one aspect, the composition is a liver organoidhaving an induced or inherent disease state similar to or identical toNAFLD, cholestasis, and/or jaundice, and said characteristic of interestis efficacy for one or a plurality of such disease states.

In one aspect, a method of identifying a donor individual in apopulation is disclosed. The method may comprise the step of creating arepresentative pooled cell composition and identifying a cell that is animmunological match to a recipient in need of cells from said donorindividual.

In one aspect, a method of identifying a genetic basis of a phenotype isdisclosed. The method may comprise the steps of contacting an organoidor differentiated cell pool comprising cells derived from a pooled cellpopulation derived from more than one donor; contacting said organoid ordifferentiated cell pool with a substance of interest, for example, adrug or drug-like substance (or any substance for which determining acellular response is desired); assaying a phenotype of interest in saidorganoid or differentiated cell pool; and correlating said phenotypewith a genotype from said pooled cell population.

In one aspect, a “Drop out assay” is disclosed. In this aspect, genomicDNA in environments (environmental DNA, or “eDNA”), a form of cell-freeDNA (cfDNA), exists in the environment to determine biodiversity such assea and soil. Detection of circulating tumor derived cfDNA is anevolving component of cancer precision medicine. Applicant found thatcells produce eDNA in a measurable amount in the cell culturesupernatant. Remarkably, when applied into pooled stem cell culture,quantitative detection of respective donor correlates with the cellratio in culture. Therefore, Applicant concluded eDNA-based noninvasivedonor identification method is effective decoding strategy for pooledstem cell culture by eDNA in culture supernatant. PCR of eDNA in culturesupernatant can be carried out using donor-specific SNP primers. EachSNP primer may be standardized, and the donor ratio of the iPSC pool canbe estimated using eDNA contained in the culture supernatant (see FIG.19). As a result, the cell ratio may be successfully determined by PCRof eDNA in the medium (see FIG. 20). In addition, these methodologiesmay be applied into differentiated progeny such as hepatocyte and liverorganoid derived from iPSC pool (see FIG. 21). In order to confirm thesensitivity of eDNA donor identification, Applicant monitored cell ratiochange by drug selection. Three donors—two fluorescently labeled donorsand one puromycin resistant donor—were mixed and cultured in a puromycincontaining media. As a result of eDNA based donor detection, puromycinresistant cells dominated over time, whereas two other donors died dueto puromycin toxicity. These results were further confirmed byfluorescent imaging in parallel, suggesting specific donor drop out canbe measurable by eDNA method, hereafter named ‘drop out assay’ (see FIG.22). Finally, as a proof-of-principle drug screening assay, hepatotoxicdrug treatment was performed using fat-treated hepatocytes prepared from11 iPSCs pool (see FIG. 23). eDNA based drop-out assay confirmed cellswith high fat accumulation risk donor were significantly more sensitiveto drug induced hepatotoxicity than healthy donor derived cells (FIG.23).

In one aspect, “Adaptive immune-matching” is disclosed. In this aspect,a pool of a representative population of iPSCs to create a “panel” canbe made. At least some of the pools may be an immunological match topotential patient (recipient). The approach may be used to waive theprior matching of stem cell to recipient, thus reducing thelabor-intensive collection of stem cell library as well as costassociated with banking.

In one aspect, a method of identifying a genetic basis of a phenotype isdisclosed. The method may comprise the steps of developing an organoidfrom pooled patients, exposing substances of interests such as drug,analyzing individual response (phenotype) in a pool, and re-analyzingoriginal genome data to match up the phenotype to know the genomiccontribution.

EXAMPLES

The following non-limiting examples are provided to further illustrateembodiments of the invention disclosed herein. It should be appreciatedby those of skill in the art that the techniques disclosed in theexamples that follow represent approaches that have been found tofunction well in the practice of the invention, and thus can beconsidered to constitute examples of modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

Synchronization of Growth Kinetic Variabilities Among Multiple iPSCs

Applicant has shown that hypoxic conditions can be used to synchronizegrowth kinetic variabilities. Hypoxic culture methods enabled successfulgrowth synchronization. Applicant has successfully identifiedsynchronizing factors including oxygen and inhibitor treatments. Forexample, cell growth in the presence and absence of an estrogen-relatedreceptor gamma agonist (GSK5182), a mitogen-activated protein kinaseinhibitor (PD0325901), a glycogen synthase kinase-3 inhibitor(CHIR99021), a Rho-associated kinase inhibitor (Y-27632) and a type 1transforming growth factor-b receptor inhibitor (A-83-01), in ambientand hypoxic conditions may be used to synchronize cells from multipledonors. In one aspect, a hypoxic culture method of 2% O2 conditions maybe used to enable successful growth synchronization and maintenance ineach cell population.

Expansion and Differentiation of Stem Cell Pool Into Specific Cell Types

Applicant has further demonstrated co-differentiation induction ofvarious cell types from iPSC pool. DE, mesoderm, posterior foregut,endothelia induction (and tube formation), hepatocyte induction, liverorganoid induction, showing that pooled cells are competent formulti-lineage differentiation, and that iPSC pooling overcomesdifferentiation propensity to endothelial cells.

An iPSC pool with a large population which may then be used to decodethe individuals at single cell level. For example, 10-15 cell lines poolmay be differentiated into liver organoids, and both genomic and cDNAmay be sequenced. Analyzed at single cell with Drop-seq (a large-scalesingle cell sequencing method), donor difference effect and rarepopulation effect may be detected. Such stem cell synchronizationapproach may provide an unprecedented powerful approach for evaluating apopulation phenotype in a dish, allowing for conducting clinical trialsin a dish and promoting drug development process as well as precisionmedicine.

“Human Organoid Trial” (HoT) in a Dish

Population pooling into an iPSC-panel, for example, Asian, American, andAfrican populations, can be pooled into an iPSC-organoid panel. Usingmulti-plexed high-content imaging, various cell parameters may bemeasured. Such parameters include, for example, cell death, cholestasis,mitochondrial overload, and ROS production. Such parameters may beassessed in response to one or more compounds of interest. The systemmay be used for drug development including efficacy, safety, transplanttherapy, and adaptive immune-matching.

Analysis of the Donor-Dependent Variation

Cell proliferation and metabolism to examine donor dependence of eachcell may be analyzed. Cell proliferation and metabolism of each iPSC maybe very different, as determined by NMR based metabolomics data and cellproliferation data.

Screening of cell synchronized condition. 1) first, growth kineticvariables may be synchronized by hypoxic culture conditions (about 2% toabout 5% O2) by regulating cell growth kinetics. 2) compound cocktailmay be used to promote iPSC synchronization. The compound cocktailtreatment may support cell synchronization by combining with the hypoxicculture. The SYNDEX (synchronization index) can be used to show thereciprocal of donor difference standard deviation.

Pooling of multi-donor iPSC. Hypoxic conditions may enablesynchronization and maintenance in each cell population on same dish.

Detection of the rare phenotype in iPSC pool. In one aspect, a rarephenotype may be detected in an iPSC pooled composition. For example,for detection of hemochromatosis, iPSCs from individual 1 and individual2 and individual 3 may be pooled and subjected to co-differentiation toform a liver organoid according to methods known thin the art. Ironaccumulation in the resulting liver organoid may be detected by liveimaging. The hemochromatosis phenotype could then be distinguished frompooled organoids in the accumulated state of iron. the hemochromatosispopulation can be distinguished from the pooled organoid. Thus, themethods may be useful for detecting the influence of a rare diseaseeffect in a pooled sample.

Maintenance of PSCs

TkDA3 human iPSC clone used in this study was kindly provided by K. Etoand H. Nakauchi. The TkDA3 human iPSC clone used in this study waskindly provided by K. Eto and H. Nakauchi. 1231A3, 317D6, 1383D6, andFF01 were gifted by Kyoto University (Japan). CW027, CW077, CW150 andWD90, 91, 92 were purchased from Coriell (N.J., USA). Human iPSC lineswere maintained as described previously (Takebe et al., 2015; Takebe etal., 2014). Undifferentiated hiPSCs were maintained on feeder-freeconditions in mTeSR1 medium (StemCell technologies, Vancouver, Canada)on plates coated with Matrigel (Corning Inc., NY, USA) at 1/30 dilutionat 37° C. in 5% CO₂. Culture oxygen conditions were performed with 2%,5% and ambient (20%) using multi-gas incubator MCO-19 MUV (Panasonic,Japan).

Inhibitor Treatment for Synchronization of PSCs

After being cultured in a 6 multi-well plate, cells were treated withinhibitors for growth synchronization. The following antibodies wereused: GSK5182 (agonist for estrogen-related receptor γ),PD0325901(mitogen-activated protein kinase inhibitor),CHIR99021(glycogen synthase kinase-3 inhibitor), Y-27632 (Rho-associatedkinase inhibitor) and A-83-01 (type 1 transforming growth factor-breceptor inhibitor).

Differentiation of PSCs into Definitive Endoderm

Differentiation of hiPSCs into definitive endoderm was induced usingpreviously described methods with slight modifications (Spence et al.,2011). In brief, colonies of hiPSCs were isolated in Accutase (ThermoFisher Scientific Inc., Waltham, Mass., USA) and 150000 cells/mL wereplated on Matrigel coated tissue culture plate (VWR Scientific Products,West Chester, Pa.). Medium was changed to RPMI 1640 medium (LifeTechnologies, Carlsbad, Calif.) containing 100 ng/mL Activin A (R&DSystems, Minneapolis, Minn.) and 50 ng/mL bone morphogenetic protein 4(BMP4; R&D Systems) at Day 1, 100 ng/mL Activin A and 0.2% fetal calfserum (FCS; Thermo Fisher Scientific Inc.) at Day 2 and 100 ng/mLActivin A and 2% FCS at Day 3. For Day 4-6, cells were cultured inAdvanced DMEM/F12 (Thermo Fisher Scientific Inc.) with B27 (LifeTechnologies) and N2 (Gibco, Rockville, Md.) containing 500 ng/mlfibroblast growth factor (FGF4; R&D Systems) and 3 μM CHIR99021(Stemgent, Cambridge, Mass., USA). Cultures for cell differentiationwere maintained at 37° C. in an atmosphere of 5% CO2/95% air and themedium was replaced every day. Differentiated definitive endoderm showedbudding on the plate at Day 7.

Freezing Posterior Foregut (pFG) Cells

Carefully aspirate media from the cell culture flask. (Rinsing with PBSis not necessary) Immediately add Accutase to the flask in an amountsufficient to cover the cells (typically 1 mL/well for a 6 well plate).Set the flask in 37° C. incubator for 5-6 min. Check the flask to see ifcells have rounded. Smack the flask against the palm of your hand todislodge any “stickers”. Gently disperse the cells. Add wash media andtake a sample to count cells. Centrifuge for 3 min @ 200-300×g. Aspiratesupernatant. Add mTESR1+Rock inhibitor (10 uM). Plate cells (2-5×10⁴ formaintenance). Passaging pFG cells. Label freezing vials with cell line,cell number, date, freezer medium, growing medium, and plate coating(laminin or Matrigel). Aspirate supernatant. Add 500 uL Cell Bankerl to0.5-2×10⁶ cells. Add vial to Nalgene Cryo Freezing container for 24 hr.Store at −80° C., or transfer to liquid nitrogen for long-term storage.

Lipids and Iron Live-Cell Imaging

After being cultured in an ultra-low attachment 6 multi-well plate, 5-10HLO were picked up and seeded in a Microslide 8 Well Glass Bottom plate(Ibidi, Wis., USA) and subjected to live-cell staining. The followingantibodies were used: BODIPY® 493/503 for lipids (Thermo FisherScientific Inc.) and FeRhoNox-1 546 for iron accumulation (GoryokagakuInc.) Nuclear staining was marked by NucBlue Live ReadyProbes Reagent(Thermo Fisher Scientific Inc.). HLO was visualized and scanned on theKEYENCE BZ-X710 Fluorescence Microscope (Japan). The final lipid dropletvolume was calculated by IMARIS8 and normalized by each organoid size.

All percentages and ratios are calculated by weight unless otherwiseindicated.

All percentages and ratios are calculated based on the total compositionunless otherwise indicated.

It should be understood that every maximum numerical limitation giventhroughout this specification includes every lower numerical limitation,as if such lower numerical limitations were expressly written herein.Every minimum numerical limitation given throughout this specificationwill include every higher numerical limitation, as if such highernumerical limitations were expressly written herein. Every numericalrange given throughout this specification will include every narrowernumerical range that falls within such broader numerical range, as ifsuch narrower numerical ranges were all expressly written herein.

The dimensions and values disclosed herein are not to be understood asbeing strictly limited to the exact numerical values recited. Instead,unless otherwise specified, each such dimension is intended to mean boththe recited value and a functionally equivalent range surrounding thatvalue. For example, a dimension disclosed as “20 mm” is intended to mean“about 20 mm.”

Every document cited herein, including any cross referenced or relatedpatent or application, is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

While particular embodiments of the present invention have beenillustrated and described, it would be obvious to those skilled in theart that various other changes and modifications can be made withoutdeparting from the spirit and scope of the invention. It is thereforeintended to cover in the appended claims all such changes andmodifications that are within the scope of this invention.

1. A composition comprising a differentiated cell population, whereinsaid differentiated cell population comprises cells from more than oneindividual.
 2. The composition of claim 1, wherein said differentiatedcell population is selected from definitive endoderm, mesoderm, exoderm,posterior foregut, endothelium, hepatocytes, or an organoid.
 3. Thecomposition of claim 1, wherein said more than one individual comprisesa population of individuals having a disease of interest.
 4. Thecomposition of claim 1, wherein said differentiated cell populationcomprises an organoid selected from a liver organoid, a gastricorganoid, an intestinal organoid, a brain organoid, a pulmonaryorganoid, a bone organoid, a cartilage organoid, a bladder organoid, ablood vessel organoid, an endocrine organoid, a sensory organoid.
 5. Amethod of making a composition comprising a differentiated cellpopulation, wherein said differentiated cell population comprises apopulation of cells derived from more than one individual, comprisingthe step of exposing a pooled-precursor cell population, to asynchronization condition until a synchronized pooled-precursor cellpopulation is formed; wherein said synchronization condition comprises amixture comprising a MEK/ERK Pathway Inhibitor, and a glycogen synthasekinase-3 (GSK3) pathway inhibitor.
 6. The method of claim 5, whereinsaid mixture further comprises an estrogen-related receptor gamma (ERRγ)agonist, a RHO/ROCK pathway inhibitor, and a type 1 transforming growthfactor-b (TGFB) receptor pathway inhibitor.
 7. The method of claim 5,wherein said synchronization condition comprises a hypoxic culturecondition between about 2% O₂ to about 5% O₂.
 8. The method of claim 5,further comprising culturing said synchronized pooled-precursor cellpopulation until definitive endoderm is formed.
 9. The method of claim8, wherein said definitive endoderm is further differentiated into anorganoid composition, wherein said organoid composition comprises cellsfrom more than one donor.
 10. A method of making a compositioncomprising a differentiated cell population from more than one donorcomprising a. exposing a precursor cell population to a clonal conditionuntil a synchronized pooled-endoderm population is formed; b. contactingsaid pooled-endoderm cell population with a mixture, wherein saidmixture comprises a TGF-beta receptor pathway inhibitor, a glycogensynthase kinase-3 (GSK3) pathway inhibitor, an FGF pathway stimulant, anEGF pathway stimulant, and a VEGF pathway stimulant; wherein one or bothof said step a and step b are carried out in a hypoxic culture conditionof between about 2% O₂ to about 5% O₂.
 11. The method of claim 10,wherein said pooled-endoderm population comprises posterior foregutcells.
 12. A method comprising contacting a test compound of interestwith the composition of claim
 1. 13. The method of claim 12, whereinsaid characteristic of interest is selected from one or more of celldeath, cell growth, cell viability, cholestasis, mitochondrial overload,ROS production indicative of mitochondrial dysfunction, fibrosis, cellstiffness, fibrosis, and efficacy for a disease state.
 14. The method ofclaim 12, wherein said test compound is selected from a drug-likemolecule, a therapeutic agent, a nutritional supplement, or combinationsthereof.
 15. The method of claim 12, wherein said composition is a liverorganoid and said characteristic of interest is efficacy forNAFLD/cholestasis/jaundice, wherein a disease state is induced in saidliver organoid.
 16. (canceled)
 17. (canceled)